**4.1. NPs promoting Ag cross-presentation for delivering cancer vaccines**

During an immune response, exogenous Ags are usually processed and presented via MHC-II by APCs to CD4+ T cells; however, tumor Ags engulfed by APCs require to be presented via MHC-I to induce production of Ag-specific CTLs, which are the main effector cells against tumor cells, thus precluding traditional methods from engineering cancer vaccines as they rely on soluble protein or peptide tumor Ags which often skew immune responses to CD4+ T cell responses while failing to induce robust CTL responses which are sufficient for cancer immunotherapy. Fortunately, it is disclosed that tumor Ags delivered by the elaborately designed NP-based multifunctional VADS able to promote lysosome escape, which is translocation of Ags from endosomes or phagosomes to cytosol avoiding Ag degradation within lysosomes, may regulate Ags to be reloaded to endoplasmic reticulum (ER)-attached MHC-I for cross-presentation and favorably elicit CD8+ T cell responses [24]. As such, to engender Ag lysosome escape, great efforts have been focused on pH-sensitive delivery systems that can retain the loaded cargo under the physiological pH condition while triggering release of Ags and disruption of endocytic vacuoles at the acidic (below pH 6) endosomal microenvironment [42], as exemplified by a pH-sensitive liposomal VADS which is formulated with a dextran derivative and was shown to promote cytosolic delivery of Ags [43].

More recently, Wang and colleagues through fabricating two types of pH-sensitive multifunctional liposomes, the mannosylated lipid A-liposomes (MLLs) and the stealth lipid A-liposomes (SLLs) both loaded with Ags and NH<sup>4</sup> HCO<sup>3</sup> , into microneedles prepared the proSLL/MLL-constituted microneedle array (proSMMA), which dissolved rapidly recovering the initial MLLs and SLLs upon rehydration [12]. Mice vaccinated with proSMMAs by vaginal mucosa patching elicited robust Ag-specific humoral as well as cellular immunity at both systemic and mucosal levels, especially, in the reproductive and intestinal ducts. Further exploration revealed that the Ags delivered by either liposomes were cross-presented for MHC-I displaying by APCs thanks to lysosome escape and reactive oxygen species stimulation, both of which occurred when lysosomal acidifying the liposome-released NH4 HCO<sup>3</sup> into CO<sup>2</sup> and NH4 + /NH<sup>3</sup> to rupture lysosomes by gas expansion and to cause ROS production by excessive ammonia induction, resulting in a mixed Th1/Th2 type response which was also promoted by liposomal lipid A via activation of TLR4, indicating the proSMMAs a multifunctional VADS capable of engendering Ag lysosome escape to elicit robust humoral and cellular immunity against Ags and a promising platform for making both cancer and infection vaccines.

In addition, an alternative approach for evading lysosome degradation of Ag includes multifunctional VADS constituting of the oxidation-sensitive polymersomes that can respond to the oxidative environment of endosomes and deliver Ags and adjuvants to cellular cytosol for induction of cellular immune responses [44]. Notably, liposomes modified with a cellpenetrating peptide octaarginine were also reported to be able to promote cross-presentation Ags and elicit production of anticancer CTLs, because the membrane-penetrating liposome enhanced proteolysis of the exogenous Ags by proteasomes and amino peptidases facilitating promoting the C-terminal trimming of antigen peptide and the production of mature MHC-I peptides [45]. Also, gold nanoparticles displaying tumor Ags were reported to enable efficient antigen delivery to dendritic cells and then activate the cells to facilitate cross-presentation, inducing Ag-specific CTL responses for effective cancer immunotherapy [46].
